The present invention relates to the activation of enzymes by their release from an immobilizing moiety, and in particular heat-mediated release.
The increasing availability of enzymes from various organisms with specific defined activities has led to the use of these catalysts as reagents in many in vitro and in vivo systems. Notably, methods of detection and analysis in the area of molecular biology require the use of a least one of the enzymes involved in DNA/RNA replication, transcription and/or translation. Precise control of the activity of these enzymes is generally achieved through precise knowledge of their pH, temperature, ionic strength and cofactor requirements and the consideration of other criteria essential for their working. Not only is the ability to control activation of these enzymes important, but also the ability to inactivate the enzymes, generally reversibly. The capacity to turn the activity of an enzyme on and off is often crucial to the correct functioning of a particular analytical or diagnostic assay.
For many years a limitation of molecular biological methods was the difficulty in obtaining sufficient amounts of homogeneous DNA for further analysis. This problem was largely overcome by the development of the Polymerase chain reaction (PCR) method which has, since its inception in the late 1980s, been responsible for many of the advancements in the genetic engineering field. A number of related amplification techniques employing the same principle as PCR have evolved from the basic concept of PCR, namely cycles of replication, denaturation and reannealing with suitable primers.
In order to obtain an amplified DNA product which is homogeneous, strict regulation of the cycles is required, in terms of time and temperature, the activity of the enzymes employed, for example the Taq polymerase, the choice of primers and the conditions of hybridization and denaturation. Thus, ideally, primers which anneal to complementary strands of target double stranded DNA are added to the DNA which has been denatured. The primers are then annealed under suitable conditions of stringency to prevent binding to non-complementary sequences. DNA extension along the length of the DNA template 3"" of the annealed primer is then performed using a suitable DNA polymerase. After a desired extended product is achieved, denaturation conditions are effected to allow separation of the parent and daughter strands which may then reenter the cycle.
In the laboratory situation, temporal control of each consecutive event is not routinely performed and a reaction mix is employed in which the change of temperature during the course of the cycle is the initiator of the consecutive steps. However, reliance on such temperature control can lead to problems resulting in a heterogeneous product. For example, in experimental procedures in which a reaction mix is heated to a temperature of around 90xc2x0 C. to effect denaturation, if all the components necessary for the polymerase reaction are present during the heating step, spurious annealing of the primers to non-complementary strands and subsequent extension may occur, resulting in amplification of non-target DNA. Although efficiencies of thermostable DNA polymerases are greatly reduced at ambient temperature relative to their peak efficiencies at higher temperatures, sufficient activity may be present at ambient temperatures to cause PCR side-products. Commonly, dimerized primer-amplified fragments (xe2x80x9cprimer dimerxe2x80x9d) as well as larger non-specific side-reaction products (mis-primed products) are obtained. The non-specific fragments can vary in size and yield, are primer sequence dependent and are most likely to arise from reactions using complex (e.g. genomic) DNA. Such non-specific fragments have been observed to reduce the yield of desired specific fragments through competition with the specific target in the reaction. Furthermore, non-specific products that are approximately the same size as the specific product can cause confusion when interpreting results. PCR amplifications particularly prone to generation of a variety of side reaction products include those involving one or more of the following: complex genomic DNA or cDNA templates; degenerate primers; very low-copy-number targets; large numbers of thermal cycles (i.e.  greater than 35); more than one target sequence in the same tube (i.e. multiplex PCR).
This has led to the introduction of a number of techniques for initiating the cycle at the temperature of denaturation, the so-called xe2x80x9chot startxe2x80x9d method (Chou et al. (1992) Nucl. Acids Res., 20, 1717-1723; D""Aquila et al. (1991) Nucl. Acids Res., 19, 3749; Faloona et al. (1990) 6th International conference on AIDS,. San Francisco, Calif., USA, Abstract No. 1019). xe2x80x9cHot startxe2x80x9d methods have found particular utility for long range PCR. For many years PCR was restricted to amplification of a few thousand bases. However, successful amplification of up to 40 kbp has been achieved using a mixture of different thermostable enzymes employing the xe2x80x9chot startxe2x80x9d procedure. If the xe2x80x9chot startxe2x80x9d procedure is not used in this case, comparatively short non-specific products are preferentially amplified. The xe2x80x9chot startxe2x80x9d procedure is also beneficial when low-copy-number targets are to be amplified or for in situ PCR.
The original approach to achieve xe2x80x9chot startxe2x80x9d of the polymerase reaction was to withhold an essential reagent of the reaction (for example the DNA polymerase, MgCl2, primers, deoxyribonucleoside triphosphates and/or DNA sample) until the reaction mixture was heated to a high temperature (e.g.  greater than 55xc2x0 C.), followed by the addition of the missing component. Another approach is the use of a heat-labile wax or jelly barrier that melts and permits mixing of aqueous components at an elevated temperature. However, both these xe2x80x9chot startxe2x80x9d methods suffer from the drawback that they have increased probability of crossover contamination on reopening the reaction tubes and that they are cumbersome and time-consuming when working with multiple samples.
An alternative to xe2x80x9chot startxe2x80x9d approaches which prevents PCR product carryover to subsequent cycles and allows the addition of all components of the PCR reaction at one time, involves the use of dUTP and the DNA repair enzyme uracil-N-glycosylase (UNG) in PCR reactions. In this method UNG digests the dU-incorporated nonspecific products before thermal cycling commences, thereby reducing the amplification of these side products in the reaction (Kwok et al. (1992) 92nd Gen. Mtg. Am. Soc. Microbiol., 116 (Abstract No. D-120). However, this method is not widely used owing to the added expense of the additional reagents and the reduced yield of specific products which may result as a consequence of using dUTP in PCR.
A xe2x80x9chot startxe2x80x9d method which allows the addition of all components of the PCR reaction at one time employs an antibody marketed by Clontech Laboratories, Palo Alto, California, USA, which binds to and inactivates Taq polymerase at ambient temperatures, but releases the active DNA polymerase once the high temperatures (above 70xc2x0 C.) have been obtained. However, not all antibodies are suitable for this methodology as it was found that of the IgGs derived from 12 hybridoma clones whose supernatants had affinity for Taq DNA polymerase, only the IgGs from 4 of the clones inactivated Taq polymerase in solution. The remainder although having affinity for the polymerase did not block its activity. Furthermore, this method suffers from the drawback that the active polymerase is released from the inactivating antibody at a particular temperature which cannot be manipulated to suit the particular requirements of different PCR reactions.
Surprisingly, it has now been found that enzymes may be reversibly inactivated by attachment to an immobilizing moiety, and activated by their release from said moiety. In particular, it has been found that Taq polymerase may be inactivated when immobilized, but may be activated by disruption of the association with the immobilizing moiety.
In one aspect, therefore, the present invention provides a method of activating a reversibly inactivated enzyme, wherein the enzyme is inactivated by attachment to an immobilizing moiety and is activated by release from said moiety.
Enzymes within the scope of the invention include any of the enzymes known and described in the art, but especially enzymes employed in the replication, transcription and translation of DNA or RNA. In particular such enzymes include polymerases, ligases, reverse transcriptases, replicases, exonucleases and ribozymes. Also included are enzyme entities which are functionally equivalent to native enzymes, but which have been modified by genetic or chemical manipulation, and which may have structural or sequence homology. Active fragments of enzymes may also be used. The invention has been shown to be particularly effective in the case of enzymes which move relative to their substrates during the course of the reaction they catalyze. Such xe2x80x9ctranslocatablexe2x80x9d enzymes include for example DNA polymerase enzymes which act on successive portions of the DNA template strand during the replication reaction. After immobilization the enzyme and its substrate (or other components of the catalytic reaction) are no longer able to interact appropriately. Whilst not wishing to be bound by theoretical considerations, one possible mechanism which may explain the inability of translocatable enzymes to function when immobilized is that the movement necessary for the correct functioning of such translocatable enzymes is hindered or prevented by immobilization. An alternative mechanism to explain the inactivation of enzymes on immobilization is that active sites available before immobilization are subsequently inaccessible. For example, if the immobilizing moiety is a hydrophobic solid support and hydrophobic regions make up the active site, binding between these regions may effectively remove the active site making it inaccessible to substrates.
Preferably, the enzymes have DNA polymerase, reverse transcriptase or ligase activity and especially preferably they are also thermostable.
As will be described in more detail below, the enzymes may be used in the form of a fusion protein comprising all or a portion of the enzyme fused with an additional polypeptide or peptide moiety. Fusion may be effected genetically or chemically using techniques well known in the art.
The immobilizing moiety bound to the enzyme may be any solid support which upon binding inhibits the activity of the enzyme in question to a level which is conducive to the requirements of the assay or system for which it is used. Numerous solid supports suitable as immobilizing moieties according to the invention, are well known in the art and widely described in the literature and generally speaking, the solid support may be any of the well-known supports or matrices which are currently widely used or proposed for immobilization, separation etc. in chemical or biochemical procedures. Thus for example, the immobilizing moieties may take the form of particles, sheets, gels, filters, membranes, microfibre strips, tubes or plates, fibres or capillaries, made for example of a polymeric material e.g. agarose, cellulose, alginate, teflon, latex or polystyrene. Biochips may be used as solid supports to provide miniature experimental systems as described for example in Nilsson et al. (Anal. Biochem. (1995), 224, 400-408) or as a diagnostic tool. Particulate materials, especially beads, are generally preferred. For example, Sepharose or polystyrene beads may be used. The immobilizing moiety may comprise magnetic particles, which permit the ready separation of immobilized material by magnetic aggregation. The requirement of such a moiety is that it is stable to the conditions at which release and hence activation of the enzyme moiety is needed.
It is also envisaged that it may be possible to design the immobilizing moiety such that on subjection to the xe2x80x9creleasexe2x80x9d conditions, although not actually physically set free from the enzyme moiety, its structural configuration is altered such that the attached enzyme moiety is able to interact appropriately with its substrate and/or other essential components. The term xe2x80x9creleasexe2x80x9d as used herein thus includes not only physical separation of the enzyme from the support but also situations where, although not physically freed from the support, the conformation of the support and/or enzyme is altered such that the enzyme may resume correct functioning. More commonly however, activation is achieved by physical separation of the enzyme from the immobilizing moiety.
The immobilizing support may carry further moieties for attachment of the enzyme. Generally speaking, these will comprise one of a pair of affinity binding partners, such as biotin and avidin or streptavidin, PNA or DNA and DNA binding protein (e.g. either the lac I repressor protein or the lac operator sequence to which it binds), antibodies (which may be mono- or polyclonal), antibody fragments or the epitopes or haptens of antibodies. In these cases, one partner of the binding pair is attached to (or is inherently part of) the immobilizing moiety and the other partner is attached to (or is inherently part of) the enzyme of interest or catalytic fragment/mutant thereof. The afore-mentioned binding moieties may be attached to the immobilizing support by methods well known in the art, which include for example, attachment-through hydroxyl, carboxyl, aldehyde or amino groups which may be provided by treating the immobilizing support to provide suitable surface coatings. U.S. Pat. No. 4,654,267 describes the introduction of many such surface coatings.
Attachment of the enzyme to the immobilizing moiety may be achieved by any reversible means which permits ready release of the enzyme at the desired conditions. Reversible as mentioned in this context indicates only that attachment of the enzyme to the support is reversible, ie. that the enzyme may be released from the support, and not that the linkage is necessarily capable of being reformed thereafter. Attachment may be, for example, via a linkage which is sensitive to heat, light, microwaves, pH, acid or proteases. Preferably, the enzyme is attached through a heat-labile attachment which permits ready release simply by raising the temperature. In such a case, once a suitable temperature is obtained in a reaction, for example in PCR, the active enzyme is released. Such attachment may for example occur through antibody binding to an epitope of the enzyme or of an appended non-functional moiety, which is disrupted by heating. Alternatively, other heat-labile protein-protein, protein-DNA or DNA-DNA attachments may be used, wherein DNA may also be replaced by PNA. For example, biotin-streptavidin, protein A-antibody, protein G-human serum albumin (HSA) pairing may be used, with either component of the pair (or functional part thereof) being attached to, or inherently part of, the enzyme moiety. For example, a synthetic monovalent IgG-binding domain Z derived from protein A or a serum-binding region BB or ABP from protein G (which are referred to in the Examples hereinafter) may be used to bind to IgG or HSA respectively. Where applicable, for example in fusion proteins, more than one of the above mentioned Z or B domains may be employed to increase binding to a solid support bearing the other component of the pair (Nilsson et al., Eur. J. Biochem. (1994), 224, 103-108). Specific antibodies and molecules bearing the epitope to which the antibody is directed may also be used, e.g. biotin:anti-biotin antibody.
Proteins or polypeptide fragments which interact with specific DNA sequences may be employed, for example proteins containing the zinc finger motif or proteins of the lac system. Of particular use may be the use of stretches of double stranded DNA in which one strand is immobilized and the other attached to the enzyme moiety. The length and sequence/GC content of the DNA stretch may be made such that the melting temperature, Tm, may be predicted and customized to the reaction in question. Thus, in PCR for example, the temperature at which the DNA polymerase is released may be strictly controlled.
Release may also be effected indirectly by heat. For example, proteinases active only at extreme temperatures or activated at extreme temperatures may be used to cleave specific cleavage sites between the enzyme and immobilization moiety.
The above-mentioned method has particular utility in PCR and analogous nucleic acid amplification techniques. As mentioned above, there is a need for an improved method of xe2x80x9chot startxe2x80x9d in nucleic acid amplification procedures, and a temperature-dependent enzyme activation method according to the present invention has particular utility in this regard.
In another aspect, the present invention thus also provides, a method of amplification of nucleic acid comprising subjecting a sample of nucleic acid to one or more cycles of in vitro amplification using an amplification enzyme, characterised in that said enzyme is provided in reversibly inactivated form attached to an immobilising moiety and is activated by release from said moiety during the first amplification cycle, whereupon the enzyme-catalyzed amplification reaction may proceed. Preferably a thermostable amplification enzyme is employed and release is effected by raising the temperature.
The generation of non-specific amplification products is thus reduced or avoided.
Any in vitro amplification method may be used, including especially PCR and its modifications.
The activation temperature necessary to achieve release of the enzyme conveniently may be the temperature to which the reaction mix is heated in the first cycle to achieve strand separation. This may be selected to suit the system and temperatures up to e.g. 90-95xc2x0 C. may be used. Alternatively, lower temperatures e.g. 70-77xc2x0 C. or down to 40-55xc2x0 C. may be more appropriate in other systems, depending on the amplification method, enzyme, reaction conditions, nature of the nucleic acid being amplified, etc.
Modifications of the classical PCR method include, for example, the use of nested primers, in which an additional two xe2x80x9cinnerxe2x80x9d primer are used, which xe2x80x9cnestxe2x80x9d or hybridise between the first xe2x80x9couterxe2x80x9d primer pair. The use of four separate priming events results in increased specificity of the amplification reaction.
Other amplification techniques worthy of mention include Ligase chain reaction (LCR), Self-sustained Sequence Replication (3SR), the Q-beta replicase amplification system and the NASBA technique (see for example Abramson and Myers (1993) Current Opinion in Biotech., 4, 41-47).
LCR which may be used to both amplify DNA and discriminate a single base mutation employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand. Thermostable DNA ligase will then covalently link each set, provided that there is complete complementarity at the junction. This concept forms the basis of the Oligonucleotide Ligation Assay (OLA) described by Landegren et al. (Science (1988), 241, 1077-1080). Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified and is therefore distinguished. The use of ligases in PCR-based methods is reviewed by Barany (PCR Methods and Applications 1991, 1, 5-16).
In 3SR, primers are used which carry RNA polymerase binding sites permitting the action of reverse transcriptase to amplify target RNA or ssDNA. In a modification of this technique, referred to as Reverse transcriptase-PCR (RT-PCR) enzymes having reverse transcriptase and DNA polymerase acivities may be used such that first strand cDNA synthesis (as achieved by the activity of reverse transcriptase) may be coupled to PCR amplification by the action of the DNA polymerase. Such enzymes include for example Thermus thermophilus DNA polymerase (rTTh).
In the Q-beta replicase system, an immobilised probe captures one strand of target DNA and is then caused to hybridise with an RNA probe which carries as a template region a tertiary structure known as MDV-1 for an RNA-directed RNA polymerase, normally Q-beta replicase.
Thus, the enzyme of the invention may be a DNA polymerase, DNA ligase, reverse transcriptase or RNA polymerase attached to an immobilizing moiety, for example Sepharose beads, via a binding pair or antibody as described previously. In the PCR reaction, for example, at ambient temperature, the DNA polymerase is inactive, but on heating during the course of the first cycle of PCR the enzyme is released and concomitantly activated. When more than one enzyme is used during the amplification reaction, ie. when more than one amplification enzyme is present, one or both of said enzymes may be inactivated by immobilization. Thus for example, in one-step RT-PCR an immobilized DNA polymerase may be employed and the reverse transcription step may be performed using mesophilic reverse transcriptases. The DNA polymerase may then be activated during the first heating cycle of PCR.
In a further aspect, the invention provides reversibly inactivated enzymes attached to an immobilizing moiety, in particular DNA polymerase attached to an immobilizing moiety.
The invention also extends to kits, comprising at least the following:
a) for PCR, an immobilized, reversibly inactivated DNA polymerase and a pair of primers which hybridise to opposite strands of the target DNA;
b) for LCR, an immobilized, reversibly inactivated DNA ligase and two pairs of primers wherein the individual primers of the pairs hybridise to adjacent stretches of DNA and each pair hybridises to complementary regions of opposite strands of the target DNA;
c) for 3SR, an immobilized, reversibly inactivated reverse transcriptase and/or RNA polymerase, a pair of primers which hybridize to opposite strands of the target DNA (or corresponding RNA) and which each have a polymerase binding site;
d) for RT-PCR, an immobilized, reversibly inactivated reverse transcriptase with DNA polymerase activity, a primer which hybridizes to the target RNA and a pair of primers which hybridize to opposite strands of the target CDNA once formed;
e) for Q-beta replicase amplification, an immobilized, reversibly inactivated RNA-directed RNA polymerase, a DNA primer and an RNA probe with a 5xe2x80x2-MDV-1 structure, the capture oligonucleotide being immobilised or permitting immobilisation.
In all the above kits, nucleotide bases will normally be supplied together with appropriate buffers.